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Annals of Botany 109: 1307– 1315, 2012
doi:10.1093/aob/mcs074, available online at www.aob.oxfordjournals.org
Peperomia leaf cell wall interface between the multiple hypodermis and
crystal-containing photosynthetic layer displays unusual pit fields
Harry T. Horner*
NanoImaging Facility, Iowa State University, Ames, IA 50011-1020, USA
* For correspondence. E-mail [email protected]
Received: 17 January 2012 Accepted: 28 February 2012 Published electronically: 25 April 2012
Key words: Chloroplasts, crystals, druses, multiple hypodermis, leaves, photosynthetic layer, Peperomia,
interface pit fields.
IN T RO DU C T IO N
Many photosynthetic organisms represented by algae, ferns,
gymnosperms and angiosperms (Arnott and Pautard, 1970;
Horner et al., 2012) normally produce solitary, multiple
and aggregated calcium oxalate crystals in the vacuoles of
certain cells that occur in different organs, if present, depending on the species. In some cases, this may be
extreme, depending on the environment (Braissant et al.,
2004). Plant crystals have been reported many times in the
literature, dating back to the late 1600s (Leeuwenhoek,
1675; McNair, 1932; Metcalfe and Chalk, 1950; Metcalfe,
1983; Prychid and Rudall, 1999). Because of their striking
appearance anatomically, especially when viewed between
crossed polarizers using light microscopy (LM), and the
fact their function is still not clearly defined, researchers
continue to seek plant systems and methodologies to help
explain what role(s) they play in a plant’s life (Franceschi
and Horner, 1980; Horner and Wagner, 1995; Franceschi
and Nakata, 2005).
Apart from some obvious examples where crystals seem to
serve as protection against predators, such as the stinging nettle
plants (Tragia; Thurston, 1976) and members of the Araceae
such as Dieffienbachia (Gardner, 1994), there seems to be
little agreement about other possible functions for the crystals
(Zindler-Frank, 1976; Ilarslan et al., 2001; Nakata, 2003;
Franceschi and Nakata, 2005). This quandary provides an opportunity to explore plant crystal function in various suitable
systems.
Since all green leaves are exposed to sunlight and contain
photosynthetic cells with chloroplasts to capture the sun’s radiation, it is reasonable to expect that certain leaf architectures
may have evolved in ways to function optimally under lowlight and water availability conditions. This is true for plant
species that inhabit understoreys where canopy foliage can
reduce light reaching the plants and where seasonal or periodic
fluctuations in availability of water occur. Crystals commonly
are found in leaves of a wide variety of species and display a
variety of forms and macropatterns that are species and taxon
specific (Lersten and Horner, 2000, 2007, 2008, 2009, 2011;
Cervantes-Martinez et al., 2005; Horner et al., 2009, 2012).
Their presence in some leaves raise the question as to
whether they have any functional significance beyond being
of taxonomic importance.
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† Background and Aims Leaves of succulent Peperomia obtusifolia (Piperaceae), and its related species, contain a
large multilayered hypodermis (epidermis) subtended by a very small single-layered photosynthetic palisade parenchyma, the latter containing spherical aggregates of crystals called druses. Each druse is in a central vacuole
surrounded by chloroplasts. All hypodermal cell walls are thin, except for thick lowermost periclinal walls associated with the upper periclinal walls of the subtending palisade cells. These thick walls display ‘quilted’ impressions (mounds) formed by many subtending palisade cells. Conspicuous depressions occur in most mounds, and
each depression contains what appear to be many plasmodesmata. These depressions are opposite similar regions
in adjacent thin palisade periclinal walls, and they can be considered special pit fields that represent thin translucent regions (‘windows’ or ‘skylights’). Druses in the vacuoles of palisade cells occur below these pit field
regions and are surrounded by conspicuous cytoplasmic chloroplasts with massive grana oriented perpendicular
to the crystals, probably providing for an efficient photosynthetic system under low-intensity light.
† Methods Leaf clearings and fractures, light microscopy and crossed polarizers, general and histochemical staining, and transmission and scanning electron microscopy were used to examine these structures.
† Key Results Druses in the vacuoles of palisade cells occur below the thin pit field regions in the wall interface,
suggesting an interesting physical relationship that could provide a pathway for light waves, filtered through the
multiple hypodermis. The light waves pass into the palisade cells and are collected and dispersed by the druses to
surrounding chloroplasts with large grana.
† Conclusions These results imply an intriguing possible efficient photosynthetic adaptation for species growing
in low-light environments, and provide an opportunity for future research on how evolution through environmental adaptation aids plants containing crystals associated with photosynthetic tissues to exist under low-light intensity and with other stresses.
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Horner — Unusual pit fields associated with photosynthetic layer in Peperomia leaves
M AT E R IA L S A ND M E T HO DS
Plants of Peperomia obtusifolia A.Dietr. (Piperaceae), growing
in the Bessey Hall rooftop greenhouse on the Iowa State
University campus, were used in this study (Fig. 1A).
Cuttings were made to increase the number of plants for use
in later studies. The uniformly green leaves were processed
in several ways to observe and understand the leaf anatomy
and ultrastructure, and the type/shape and location of the
crystals.
Leaf clearings
Circular leaf punches using a small-diameter cork borer were
made and placed in a multiple compartment tray (Horner and
Arnott, 1961), and processed according to Lersten and Horner
(2011) to remove all cellular contents except the cell walls
and inorganic crystals. The cleared punches were mounted on
slides in Permount (www.fishersci.com), coverslipped and
viewed between crossed polarizers using an Olympus BX40
compound light microscope (www.olympus.com) fitted with a
Zeiss Axioplan colour MRc digital camera (www.zeiss.com).
Vibratome and paraffin sections
Fresh leaf segments were cut into 45 mm thick sections with a
vibratome (tpi-3000; www.tedpella.com). They were mounted
in deionized water on slides, coverslipped and immediately
viewed and photographed with the same microscopic system.
Leaf punches were also fixed in formalin – acetic acid–
ethanol (FAA; Ruzin, 1999) for 24 h at room temperature.
These punches were dehydrated through an ethanol series
(25, 50, 70, 95, 100 and 100 %), transferred to xylene and infiltrated and embedded with Paraplast paraffin (54 – 56 8C mp;
www.fishersci.com). The 10 mm thick cross- and paradermal
sections were cut with a steel knife, and mounted on glass
slides. Sections were deparaffinized and brought to water
where they were stained either with aqueous 1 % chlorozol
black E (for general contrast enhancement) or with the
aqueous periodic acid – Schiff (PAS) technique (Ruzin, 1999)
(for contrast enhancement of water-insoluble polysaccharides).
Sections were dehydrated through an ethanol series to xylene;
Permount was added along with coverslips. Sections were
viewed and photographed with the same light microscopic
system.
Scanning electron microscopy (SEM)
Leaf punches fixed in FAA for 24 h were dehydrated from 50
% ethanol through 100 % ethanol: some punches were placed in
Parafilm (www.fishersci.com) pillows filled with 100 % ethanol,
sealed, frozen in liquid nitrogen and fractured in cross-section.
Fractured pieces were placed back into 100 % ethanol. They
were treated twice more with ultrapure 100 % ethanol and critical point dried (Denton; www.azom.com) using liquid carbon
dioxide. Whole punches were sandwiched between two SEM
aluminum stubs that had double-sided sticky taped end surfaces;
the stubs were pressed together and split apart so that each stub
contained a somewhat paradermal and complementary portion
of the torn leaf punch. The fractured punches were mounted vertically on aluminium stubs, fracture side up. Silver paint was
applied around the torn leaf punches and at the bases of the fractured leaf punches. All mounted specimens were sputter coated
(Denton; www.azom.com) with about 10 nm of palladium:gold
(80:20). Specimens were viewed and imaged with a JEOL 5800
scanning electron microscope (www.jeol.com) at 15 kV.
Transmission electron microscopy (TEM)
Small, 1-mm diameter leaf punches were fixed with 2 % paraformaldehyde/2 % glutaraldehyde in a 0.1 M cacodylate buffer
( pH 7.24) at 4 8C for 24 h. The punches were buffer washed
three times for a total of 1 h, post-fixed in 1 % osmium tetroxide
in the same buffer for 1 h, dehydrated through an ethanol series
to ultrapure ethanol and embedded in Spurr’s resin (hard).
Diamond knife-cut sections 60 – 90 nm thick were placed on
copper grids and post-stained with lead and uranium. A JEOL
1200 transmission electron microscope (www.jeol.com) was
used to view and capture the images on DuPont Cronar
high-resolution film. Processed negatives were tiff digitized.
All digitized images were processed in Adobe PhotoShop CS5
and collaged in Adobe Illustrator CS5 (www.adobe.com).
R E S U LT S
Mature P. obtusifolia leaves are succulent and measure .1 mm
in thickness (Fig. 1B). They consist of two single-layered
epidermises (upper, adaxial; and lower, abaxial), a multilayered
hypodermis (i.e. adaxial multiple epidermis by other authors)
below the upper epidermis, a single small subtending palisade
parenchyma (i.e. median mesophyll by other authors)
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Two closely related genera, Peperomia and Piper
(Piperaceae), share similar pantropical geographic ranges and
often grow under low-light conditions with variable water availability. The leaf anatomy of these two genera, however, is significantly different from each other but both contain various
types of crystals associated with their leaf chlorenchyma
tissues (Christensen-Dean and Moore, 1993; Horner et al.,
2009, 2012). With respect to the genus Peperomia, several
studies have suggested a light gathering and reflection role for
the crystals in its leaves (Schüroff, 1908; Franceschi and
Horner, 1980; Kuo-Haung et al., 2007; Horner et al., 2009),
and for some Piper species (Horner et al., 2012). Apart from
the studies of Kuo-Huang et al. (2007) on Peperomia glabella
and of Gibeaut and Thomson (1989a) on Peperomia obtusifolia
leaves, there has been no additional information regarding the
anatomical and ultrastructural features of crystal-containing palisade parenchyma cells and associated chloroplasts in other taxa
(Lichtenthaler et al., 1981). This latter feature, coupled with
these species having been identified with different photosynthetic mechanisms [C3, crassulacean acid metabolism (CAM)
and/or CAM-cycling) (Virzo de Santo et al., 1983; Ting,
1985; Sipes and Ting, 1985; Nishio and Ting, 1987; Ting
et al., 1994), suggests that further research is warranted.
Therefore, the purpose of this study is to provide a closer look
at the interface relationship between the multilayered hypodermis and palisade parenchyma in P. obtusifolia as the basis for
additional studies dealing with crystal-plastid involvement in
plants growing under low-intensity conditions.
Horner — Unusual pit fields associated with photosynthetic layer in Peperomia leaves
B
A
E
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UE
Piperaceae
Peperomia obtusifoli
a
H
VB
PP
SP
LE
D
E
F
G
H
F I G . 1. Peperomia obtusifolia whole plant, and light (LM) and scanning (SEM) electron micrographs of portions of leaves. (A) Greenhouse plant. (B) SEM
cross-section fracture showing different tissues (UE, upper, adaxial epidermis; H, multiple-layered hypodermis; PP, single-layered palisade parenchyma; SP, multiple layered spongy parenchyma; LE, lower, abaxial epidermis; VB, vascular bundle). (C) LM paradermal view of clearing between crossed polarizers focused at
the palisade parenchyma layer containing druses. Some druses are out of focus due to non-planar palisade parenchyma. (D) LM paraffin cross-section viewed
between partially crossed polarizers accentuating druses in a non-planar palisade parenchyma. Note that all but one druse are located near the common wall with
the hypodermis. (E) SEM cross-section fracture of palisade parenchyma showing its interface with the hypodermis. The arrow identifies one visible druse in a
palisade parenchyma cell vacuole. (F) SEM face-on view of the hypodermal wall common with the palisade parenchyma. Mounds represent contacts with subtending palisade parenchyma. The majority of mounds have one or more pits. (G) SEM of one pit showing blebs purported to be severed plasmodesmata. (H)
SEM cross-section fracture through a single pit. The thick hypodermal wall is identified with a vertical orange bar, and the much thinner palisade parenchyma
wall is identified with a vertical green bar. Scale bars: (A) ¼ 5 cm; (B) ¼ 250 mm; (C) ¼ 50 mm; (D, E) ¼ 25 mm; (F) ¼ 10 mm; and (G, H) ¼ 2 mm.
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C
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Horner — Unusual pit fields associated with photosynthetic layer in Peperomia leaves
altogether, exposing this upper wall and thin pit fields with
protrusions. The latter are interpreted as plasmodesmata remnants (Fig. 2F, G) similar to those observed in the basal hypodermal wall pit fields opposite them.
Oblique (almost paradermal) SEM fractures through the
hypodermal – palisade parenchyma wall interface show the
hypodermal wall mounds and the subtending palisade parenchyma cells and their druses (Fig. 2H). The druses are about
the same diameter as measured in both LM and SEM
images (average diameter ¼ 8.4 mm). They are basically
spherical in shape and consist of many individual crystals
each with several visible facets (Fig. 2I). These crystals are
bound together by an organic nucleation centre (not shown;
Horner and Wagner, 1980, 1995). The druses are composed
of the elements calcium and oxygen, as determined by X-ray
energy dispersive analysis with SEM, and they are not
subject to dissolution with 5 % acetic acid (H.T. Horner,
unpubl. res.). Based on druses analysed in this way in other
Peperomia and non-Peperomia species, the crystal aggregates
are deemed to be calcium oxalate.
The palisade parenchyma, consisting of a single layer, is
made up of ‘U’-shaped cells that are on average about
13 mm in diameter and 32 mm in length. Given the average
diameters of the basal hypodermal cells (100 mm) and palisade
parenchyma cells (13 mm), and taking into account palisade
parenchyma cell wall thicknesses and intercellular spaces, a
single hypodermal cell could be in contact with as many as
50– 60 palisade parenchyma cells.
The top of each palisade parenchyma cell is slightly
arched, whereas the base of each cell is rounded, as
shown by the fractured exposed protoplasts (Fig. 2J).
Longitudinal fractures and sections show each protoplast
consisting of a central vacuole, and a peripheral cytoplasm
containing mitochondria, peroxisomes, nuclei (not shown)
and conspicuous, lens-shaped chloroplasts with massive
grana stacks (Fig. 3A). The plastids measure 5– 6 mm in
length and several micrometres in width (Fig. 3A). Their
photosynthetic thylakoid membranes, the grana stacks, are
unusually large and sometimes contain as many as 100 thylakoids per stack (Fig. 3B). The grana stacks are typically
oriented perpendicular to the vacuole tonoplast and the
druse.
All methods of observation used show one druse in each
palisade parenchyma cell (Fig. 3C); rarely is there more than
one druse per cell (Fig. 3D). The position of the druse
within the vacuole varies depending on the individual cell,
as observed in paraffin sections and leaf fractures (Figs 1D,
2J and 3C, D).
Slightly oblique paradermal leaf sections stained with
PAS show that hypodermal cells have very few plastids
with starch grains (Fig. 3E). The palisade parenchyma
cells with their conspicuous chloroplasts have more starch
grains, and the spongy parenchyma cells immediately
below the palisade parenchyma have plastids with much
larger and more numerous starch grains (Fig. 3E). The
size of the starch grains decreases in spongy parenchyma
below this region toward the lower epidermis, and they
are smallest in the abaxial epidermis and its guard cells
(not shown).
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immediately below this hypodermis and a multilayered spongy
parenchyma (i.e. lower mesophyll by other authors) in which the
venation resides (Fig. 1B). Cleared and paraffin- and vibratomesectioned leaves show that calcium oxalate crystal aggregates,
called druses, occur in the typically single-layered palisade parenchyma (Fig. 1C), normally one druse per cell throughout the
lamina. This single layer undulates slightly so that it is not
planar but follows the bases of the lowermost hypodermal
cells (Fig. 1D, E). There are no other crystals in the leaves of
this species so it has a crystal macropattern designated as
DU – /– (D, druse; U, uniform layer of druses; – /– , neither
prisms nor raphides are present in spongy parenchyma) by
Horner et al. (2009, 2012). This crystal macropattern is predominant for the genus Peperomia. The other two major druse
macropatterns represented in the genus are: species with larger
druses over the veins (DUVbig) and smaller druses in the
lamina or areole (Asmall) regions (DUVbigAsmall – /– ); and
druses only over the veins (DR – /– ) (Horner et al., 2009,
2012). There are variations of these three macropatterns.
The purpose of this study was to focus on the interface
region between the lowermost hypodermal cells and the singlelayered palisade parenchyma, containing the druses, that subtends it and is physically attached to it. Figure 1B provides
an overview of this region that is relatively small, compared
with the remainder of the leaf anatomy.
The FAA fixation provides relatively well-preserved structures except for the hypodermal protoplasts that have extremely large vacuoles ( probably .80 % of the cell volume). The
basal hypodermal cells in paradermal section are somewhat
flattened or slightly curved, and vary in size, measuring
about 100 mm across at their bases. The hypodermal protoplasts are severely plasmolysed due to their tonicity and the
FAA fixative used, but, as such, expose the basal walls for
direct observation of their inner wall surfaces (Fig. 1F). Each
large basal hypodermal cell wall is in direct contact with
many smaller palisade parenchyma cells (see later) that are
delineated as ‘mounds’ in these basal walls (Fig. 1F). The majority of hypodermal wall mounds have one or more obvious
depressions or pits of different sizes (Fig. 1G). The depths
of the pits measure about 0.6 mm (Fig. 1H), whereas the adjacent palisade parenchyma wall is only about 0.1 mm thick. In
each pit there are what appear to be membrane-like protrusions
that seem to be severed plasmodesmata connecting the basal
hypodermal cell with the adjacent subtending palisade cell
(Fig. 1G).
In paradermal paraffin sections these mounds are clearly
stained and the pit field regions in each mound are either not
stained or only lightly stained. This is verified when using
either chlorozol black E (Fig. 2A) or PAS (Fig. 2B) staining
procedures, suggesting that only the middle lamella is
present and serves as the partition between them. The size
and number of pit fields vary per mound, and the majority
of mounds display pits (Fig 2C). These same results are
observed in three other greenhouse Peperomia species
observed in similar sections (not shown; P. clusifolia,
P. prostrata and P. subpeltata).
When fractures of the wall interface are observed from the
palisade parenchyma side in regions where the palisade parenchyma protoplasts are missing, either remnants of the protoplast plasmalemma remain (Fig. 2D, E) or they are missing
Horner — Unusual pit fields associated with photosynthetic layer in Peperomia leaves
A
C
E
F
G
H
I
J
F I G . 2. Peperomia obtusifolia light (LM) and scanning (SEM) electron micrographs of portions of leaves. (A) LM paraffin paradermal section through the hypodermal–palisade parenchyma region stained with chlorozol black E and partially crossed polarizers. Three distinct regions of mounds are visible with their pit
fields. Druses are evident in some cells of palisade parenchyma. (B) LM paraffin section comparable with A in bright-field mode, and stained with the PAS
technique. Mound regions have distinct pit fields, and subtending palisade parenchyma shows many starch grains associated with plastids. The acidic staining
method removes all crystals. (C) LM higher magnification on one interface region showing mounds with multiple pit fields. (D) SEM fracture showing the underside view of palisade parenchyma where most protoplasts are missing but membrane fragments remain at most interface regions. (E) SEM higher magnification of
the interface region with a portion of cell membrane still attached. (F) SEM interface region minus cell membrane showing a pit field from the palisade parenchyma side. (G) SEM higher magnification of a pit field showing remnants of plasmodesmata. (H) SEM oblique fracture through the interface region showing
hypodermal wall mounds with pit fields and subtending palisade parenchyma with druses. (I) SEM single exposed druse dislodged from a palisade parenchyma
cell. Many facets are visible, most with obvious twin planes. (J) SEM cross-section fracture through a palisade parenchyma cell showing peripheral chloroplasts
surrounding a druse in the middle of a vacuole. Scale bars: (A, B) ¼ 50 mm; (C, D, H) ¼ 20 mm; (E, J) ¼ 10 mm; (F) ¼ 5 mm; and (G, I) ¼ 2 mm.
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D
B
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Horner — Unusual pit fields associated with photosynthetic layer in Peperomia leaves
B
A
C
E
D
DISCUSSION
The pantropical, species-rich, basal-angiosperm genus Peperomia,
containing about 1600 species (Samain et al., 2009), are
typically small in stature, and grow mainly epiphytically, and
sometimes terrestrially, often under low-intensity light in
moist as well as sometimes dry conditions at lower or middle elevations, and to a lesser degree at higher elevations (Kaul, 1977).
The leaves of these species vary widely in their size, colour,
shape, succulence and thickness, but all appear to have a basic
leaf anatomy that includes a multiple layered hypodermis
(adaxial multiple epidermis) (Dahlstedt, 1900; Yuncker and
Gray, 1934; Murty, 1960; Datta and Dasgupta, 1977; Kaul,
1977; Takemori et al., 2003; Souza et al., 2004) subtended by
a typically single-layered palisade parenchyma (median mesophyll) that consistently contains druses composed of calcium
oxalate (Schüroff, 1908; Franceschi and Horner, 1980;
Kuo-Haung et al., 2007; Horner et al., 2009, 2012). Below the
palisade parenchyma is the spongy parenchyma. This
anatomical arrangement is characteristic for P. obtusifolia
leaves that are .1 mm thick, glabrous and shiny on the
adaxial surface, and glabrous, dull and with stomates on the
abaxial surface.
This leaf anatomy in P. obtusifolia, shows that 75 % of the leaf
volume consists of the multiple hypodermis whereas only 4 %
consists of palisade parenchyma (median mesophyll; Gibeaut
and Thomson, 1989a, b). The spongy mesophyll makes up
about 18 % of the leaf volume (Gibeaut and Thomson,
1989b). Large variations in the percentages occur between the
multiple hypodermis and the spongy parenchyma in other
species, while the palisade parenchyma remains about the
same percentage. The palisade parenchyma is considered to
be the main photosynthetic tissue in all Peperomia leaves in
the literature and has the majority of chlorophyll. In the genus
as a whole, it is the only tissue that contains the druses
(Horner et al., 2009, 2012) and chloroplasts with large grana
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F I G . 3. Peperomia obtusifolia light (LM), scanning (SEM) and transmission (TEM) electron micrographs of portions of leaves. (A) TEM of a single palisade
parenchyma chloroplast sandwiched between the cell wall and vacuole; note the extensive stacks of grana thylakoids oriented perpendicular to the vacuole (and
crystal, not shown). (B) TEM showing a granum made up of many thylakoids. (C) SEM cross-section fracture through a palisade parenchyma cell showing peripheral chloroplasts surrounding two druses in the upper part of a vacuole; chloroplasts are large. (D) SEM cross-section fracture through a palisade parenchyma
cell showing peripheral chloroplasts surrounding a druse in the middle of a vacuole. (E) LM paraffin oblique section in bright-field mode, and stained with the
PAS method. The section shows the lowermost hypodermal layer (upper right), interface, palisade parenchyma (middle) and portion of spongy parenchyma
(lower region). There are few starch grains in the hypodermis, many small ones in palisade parenchyma and much larger ones in spongy parenchyma. Starch
grains are smaller in spongy parenchyma closer to the lower (abaxial) epidermis (not shown). Scale bars: (E) ¼ 50 mm; (C, D) ¼ 5 mm; (A) ¼ 1 mm; and
(B) ¼ 0.5 mm.
Horner — Unusual pit fields associated with photosynthetic layer in Peperomia leaves
positions in cell vacuoles) from static images and cinematography suggests that the crystals, surrounded by chloroplasts,
appear to be light sensitive and thus may play a role in light
collection and dispersion for photosynthesis. The mechanism
for such movements is unknown.
Holthe et al. (1992) and Kuo-Huang et al. (2007) present data
and interpretations of studies showing that species of Peperomia
have three photosynthetic mechanisms: C3, CAM and
CAM-cycling. The latter two mechanisms are common in
some families with epiphytes and they are thought to be ways
to improve photosynthetic efficiency by allowing light to
reach the C3 photosynthetic tissues, and as a physiological adaptation to drought or water stress (Ting, 1985); both conditions
occur in Peperomia. In their study dealing with 93 Peperomia
species, Holthe et al. (1992) found that 45 (48.4 %) had C3
photosynthesis, 18 (19.4 %) had solely CAM and 30 (32.2 %)
had CAM-cycling. Ting et al. (1994) used tissue printing to
detect proteins and RNA associated with these photosynthetic
mechanisms in the multiple hypodermis, and photosynthetic
palisade and spongy parenchymas. In CAM and CAM-cycling
species, CAM occurs predominantly in the multiple hypodermis
and spongy parenchyma, whereas C3 photosynthesis is primarily limited to the palisade parenchyma. In their study, Helliker
and Martin (1997) considered P. obtusifolia to have
CAM-cycling that includes C3 photosynthesis.
F I G . 4. Diagram of a Peperomia obtusifolia palisade parenchyma cell
showing the interface of the common wall with the hypodermis containing
pit fields, and a protoplast with a large central vacuole containing a multifaceted druse surrounded by chloroplasts with large grana oriented perpendicular to
the druse. Visible light waves are shown filtering through hypodermal pit
fields, striking druse facets and dispersed to surrounding chloroplasts.
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stacks (Gibeaut and Thomson, 1989a; Kuo-Huang et al., 2007;
this study).
The P. obtusifolia multiple hypodermis is basically a
water-containing (Wasser Zellen: Haberlandt, 1904;
Schürhoff, 1908) tissue that most authors call a ‘multilayered window tissue’ immediately above the photosynthetic
palisade parenchyma. The present study shows the interface
between these two tissues to consist of a complex, relatively
thick, aggregate double wall with large, thin pit fields containing connecting plasmodesmata. These pit fields are probably mainly middle lamella, and more than likely serve as
additional skylights or windows allowing light waves filtering
through the multiple hypodermis to enter into the photosynthetic palisade layer. This vast photosynthetic network of
cells containing multifaceted druses is able to collect and
disperse the light waves to the surrounding chloroplasts
with exceptionally large grana.
Kuo-Huang et al. (2007), working with P. glabella, showed
that plants growing under lower light intensities had higher
photosynthetic rates and larger druses than plants growing at
higher light intensities. They also showed that, at lower light
intensities, the vacuolar druses were larger and nearer the
base of the palisade cells, whereas at higher light intensities
they were smaller and near the top of the vacuoles, closer to
the hypodermal – palisade parenchyma interface; and the plastids had modified thylakoids. In unpublished results (H.T.
Horner), normally appearing Peperomia druses were present
under lower light intensity conditions, and at high-light intensities the druses became partially dismantled and mis-shapen.
Therefore, it seems from the study by Kuo-Huang et al. (2007)
that light intensity has a major impact on the location, size and
shape of the druses, as well as the location and ultrastructure of
the chloroplasts. The presence of large stacks of grana and
their perpendicular orientation to the vacuolar druses (this
study) also suggest an optimization for light gathering and
photosynthetic efficiency at low-light intensities. When light
intensity is too high, movements of the plastids and druses
seem to change in ways to protect the system against photooxidation. It is more than likely that the special pit fields in
the interface region do not control the passage of light waves
but have evolved to serve only to provide an efficient means
for light to reach druses and the chloroplasts when conditions
are right. Observing this region in other species normally
growing under low- and higher light conditions should help
to determine the consistency and significance, if any, of the
special pit fields.
Horner et al. (2012), using bright-field microcinematography with fresh vibratome sections of three Piper species,
observed the vacuole crystal sand in chlorenchyma cells actively to tumble. Similar sections of P. obtusifolia were
viewed in the same way, but no movement of the palisade parenchyma druses was noted, even though images of fixed tissue
showed druses in different locations within the vacuole, as
noted by Kuo-Haung et al. (2007) and in this study. Horner
et al. (2012) suggested that in vibratome cross-sections, the
Peperomia palisade parenchyma were perpendicular to their
normal orientation in the leaf and, therefore, would not move
in this light path. With Piper crystal sand, all orientations
are equal and thus any orientation could stimulate the crystals
to move. In both taxa, ‘movement’ (as seen by different
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Horner — Unusual pit fields associated with photosynthetic layer in Peperomia leaves
AC KN OW LED GEMEN T S
I want to thank the Department of Genetics, Development and
Cell Biology for partial support of this research, Mr Steve
Mahoney, greenhouse manager of the Richard Pohl
Conservatory (on top of Bessey Hall), for providing the plant
material, and the Microscopy and NanoImaging Facility for research space and use of its instrumentation.
L I T E R AT U R E CI T E D
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It was of interest to compare the list of 93 Peperomia species
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These two characters, along with their leaf anatomy, may
have evolved independently but together to allow the genus
as a whole to cope successfully with their specialized
environments.
The combination of low-light intensity, periods of water
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and the wall interface with unusual pit fields between these
two layers, provides a unique opportunity to pursue the possible function of this integrated system further (Schüroff,
1908; Franceschi and Horner, 1980; Kuo-Huang et al.,
2007). The present study is the first step in describing the
spatial, anatomical and ultrastructural characteristics of this
wall interface (Fig. 4) and provides a ‘window’ of opportunity
to develop future studies to answer questions remaining about
its unique and biologically intriguing properties.
Horner — Unusual pit fields associated with photosynthetic layer in Peperomia leaves
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